Method and apparatus for cultivating a cell or tissue

ABSTRACT

The invention provides a method of and an apparatus for cultivating a cell or tissue capable of preventing the cell or tissue from being contaminated and realizing an efficient in vitro culture. The method of and the apparatus for cultivating a cell or tissue comprise installing a culture position (culture chamber) under an environment that is arbitrarily controlled such as an environment mimicking the living body, supplying a culture medium to the cell or tissue while the cell or tissue is held at the culture position, and cultivating the cell or tissue at the culture position that is under the ideal environment, thereby preventing the cell or tissue from being contaminated and realizing an efficient in vitro culture.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a culture technology of a cell or tissueemployed by a tissue engineering that is applied to a cell or tissueengineering or genetic treatment, particularly to a method of and anapparatus for cultivating a cell or tissue (hereinafter referred to asculture method and culture apparatus) for use in an in vitro culture ofa cell or tissue that is needed for restoring a damaged tissue of humanbody.

2. Description of the Related Art

There are following methods for restoring a damaged tissue or apathogenic part of a living body. The first method is to substitute thedamaged tissue or pathogenic part for materials other than a living bodysuch as plastic, metal, ceramic as restoring means of the damaged tissueor the pathogenic part. As substitutable materials, there are ceramicsand stainless steel for bones, a polyethylene resin for joints, and avinyl resin for blood vessels. Second method is to substitute thedamaged tissue or pathogenic part for parts of other animals or thedifferent position of the living body. As the substitutable tissue inthe second method, there are, for example, skins. The third method is totransplant of internal organs of other people.

In the first method, there is a drawback that the materials other thanthe living body such as plastic, metal, ceramic need to be substitutedperiodically by others when they are worn or consumed or materialsseparated from the materials other than the living body by the wearthereof affects adversely on the living body. Further, in a blood vesselmade of a synthetic polymer, there is a report that an interior of theblood vessel is clogged when it is used for a long period of time. Inthe third method, if there is no donor for supplies his or her internalorgans to be transplanted, it is impossible to carry out the thirdmethod. Even if the third method is carried out, there still remains aproblem of immunological rejection between internal organs of twopeople.

Accordingly, a method of restoring a damaged tissue or a pathogenic partof a living body that is expected to be carried out is to substitute thedamaged part of a cell or tissue by a cell or tissue that is obtained bycultivating a cell or tissue in vivo or in vitro. It is reported incurrent researches that there is a possibility in many tissues such asskins, cartilage, bones, blood vessels, livers, and pancreas. If a cellor tissue derived from a living body is cultivated inside or outside theliving body of a patient, and the cell or tissue obtained by the cultureis applied to the restoration of a damaged part, a tissue can beregenerated in the body, and further the tissue applied to therestoration would have gene of the patient per se, there does not occurimmunological rejection, and further, a chemical substance such assynthetic polymer other than a living material does not adversely affecta living body, thereby realizing an ideal treatment.

There has been proposed and disclosed as a technology of this type inJapanese Patent Laid-Open Publication No. 9-313166 entitled “DEVICE FORCULTURING CELL”. This technology needs to disassemble into each partevery culture, to clean, to sterilize, and then reassemble theapparatus, resulting in a risk of contamination by bacteria aftersterilization. Although each part of the apparatus can be assembled forpreventing contamination by bacteria so as to perform a sterilizationtreatment by an autoclave (absolute pressure 2 atm. at 121° C.), thistechnology can not be employed in view of the contamination by bacteriabecause a pump and a pressure sensor include many electronic devices, aspecific resin and oil. Accordingly, parts of the pump and pressuresensor are disassembled while only a passage through which a culturemedium passes is taken out and is sterilized by chemicals, and otherparts are sterilized by the autoclave, thereafter the pump and pressuresensor are assembled together with the apparatus, resulting in muchlabor and the increase of risk of contamination by various bacteria.Further, in the culture using an incubator (culture vessel), a pump or acontroller is subjected to an adverse affection by a temperature orhumidity, and also all the devices can not be accommodated in theincubator having a limited capacity. Accordingly, it is necessary toassemble the culture apparatus in a state where the incubatorcommunicates with an open air for allowing piping, a power supply and acontrolling electric wire to pass through a through hole of theincubator. Still further, since a pressure is applied to an entirecircuit of a culture medium, the entire culture apparatus includingparts of the pump and piping shall have a pressure resistantconstruction. As a result, it is very difficult to place the apparatusat high pressure e.g. not less than 1 MPa, and even if a high pressureis applied to the apparatus, the apparatus shall be high pressureresistant as a whole, resulting in a problem of high cost.

More still further, there is a research reported by Dr. Shuichi MIZUNOet al. in Harvard Medical School that a tissue of a living body iscultivated by applying a pressure to the living body as physicalstimulation (see Materials Science and Engineering C6 (1998) 301-306).According to this research, a culture apparatus is formed as illustratedin FIG. 26. Each constituent and function thereof in this cultureapparatus is described now.

A pump 400 has a role to circulate a culture medium 402 therein and topressurize the interior of a culture chamber 404 to supply a hydraulicpressure to a cell 406 or tissue, and it is formed of a pump for use ina liquid chromatograph, and further it has a control unit built thereinfor flowing a given amount of fluid.

A back pressure regulator 408 allows a pressure to escape through avalve 410 by opening the valve 410 when a pressure exceeds a pressure tobe applied to the cell 406 or tissue exceeds so as to hold the pressureinside the culture chamber 404 constant. The back pressure regulator 408is selectively provided in a circuit 426, described later, depending ona pressure to be applied to the cell 406.

The culture chamber 404 forms a space for cultivating the cell 406 ortissue, and a scaffold 412 formed of a sponge made of a collagen inwhich the cell 406 or tissue is transplanted is accommodated in thespace. The cell 406 or tissue grows on the scaffold 412 formed of asponge made of a collagen. A pressure sensor 414 detects a pressureinside the culture chamber 404 while a pressure monitor 416 indicatesthe pressure detected by the pressure sensor 414. The pump 400 iscontrolled by the pressure detected by the pressure sensor 414 and itstops its operation when the detected pressure increases to a largeextent.

A culture medium tank 418 stores therein the culture medium 402 adaptedfor the cell 406 or tissue to be cultivated and the culture medium 402comprises e.g., amino acids, saccharides, salts, and so forth. Theculture medium tank 418 communicates with an open air through a venttube 422 that penetrates a closed stopper 420, and a vent filter 424prevents the culture medium 402 from being contaminated by an open air.

The culture apparatus is accommodated in an incubator forming ahermetically sealed space. The incubator is a space for forming apleasant cultivating atmosphere and it is maintained under the optimumtemperature, humidity and gas concentration (oxygen and carbon dioxide)that is optimized for the cell or tissue. The culture medium 402 isfilled in the circuit 426 by the pump 400 and circulated therein. Theoxygen and carbon dioxide are soluble in the culture medium 402 afterthey pass through the vent filter 424, and the culture medium 402 iskept under appropriate oxygen concentration and carbon dioxideconcentration. When the pump 400 is operated, a pressure inside theculture chamber 404 gradually increases. When the pressure exceeds agiven value set by the back pressure regulator 408, the valve 410 of theback pressure regulator 408 is opened to discharge the culture medium402 so that a pressure of the culture medium 402 is decreased by theamount of the discharged culture medium 402, thereby shutting the valve410. With the repetition of these operations, a fixed pressure ismaintained, and at the same time a fixed amount of the culture medium402 is repetitively circulated. The cell 406 or tissue grows while it issubjected to such pressure application stimulation.

Although a fixed pressure is maintained in this culture apparatus, theincrease and decrease of a pressure can not be repeated. Since theincrease of the pressure is made by the pump 400, the rate of increaseof the pressure is determined by the capacity of the pump 400. If theamount of circulation of the culture medium 402 increases, the rate ofincrease of the pressure becomes fast, while if the amount ofcirculation of the culture medium 402 decreases, the rate of increase ofthe pressure becomes slow. Accordingly, if a pressure cycle iscontinuously repeated, there is a method of providing a bypass 432having a bypass valve 428 and an orifice valve (needle valve) 430 inparallel with a back pressure regulator 408 as shown in FIG. 27 todecrease the pressure. In this method, although the pressure can bedecreased, there is a drawback that it takes a long time for one cycle,and the setting of a repetitive cycle and the circulation amount of theculture medium 402 can not be independent from each other, and furtherthe regulation of the orifice valve 430 is finely controlled so as torender the rate of decrease of the pressure unstable.

Since the culture apparatus has to be assembled after each componentthereof is disassembled, cleaned and sterilized every performance ofculture, there is the risk of contamination by the bacteria aftersterilization. Although it is possible to subject the assembled cultureapparatus to a sterilization treatment by an autoclave (absolutepressure 2 atm. at 121° C.), the pump and pressure sensor can not besterilized because they include many electronic devices, specific resinand oil. Accordingly, under the existing circumstances, only a passagethrough which the culture medium 402 passes is taken out while each partof the pump and pressure sensor is disassembled, and each part issubjected to a sterilization by chemicals. The other parts of the pumpand pressure sensor are subjected to a sterilization by an autoclave,then the pump, pressure sensor and the culture apparatus arerespectively assembled, resulting in much labor and a risk ofcontamination by various bacteria.

Although oxygen and carbon dioxide are taken in the culture medium 402through a filter, they are directly taken in from an ambient atmosphere,resulting in a risk of the contamination by bacteria. Further, althoughthe culture apparatus is accommodated in an incubator, a pump unit and apressure monitor is susceptible to a temperature and humidity so thatthe pump unit and pressure monitor are hardly accommodated in theincubator in view of their capacities. Accordingly, it is necessary toassemble the culture apparatus by allowing a tube for piping, a powersupply and an electric wire for controlling to pass through a throughhole of the incubator so that the inside and outside thereof areconnected to each other.

Since a pressure is set by selecting a back pressure regulator dependingon a set pressure, when the setting of a pressure is changed, the backpressure regulator is replaced by another one, resulting in much laborand a risk of contamination by various bacteria.

When a pressure cycle is changed, a pressure at a low pressure side cannot be set in the culture apparatus in FIG. 27, but the pressure can beregulated by the orifice valve 430 so that the set pressure is varied bythe quantity of circulated flow through the pump 400 even if a pressurecan be regulated to some extent by the orifice valve 430.

As set forth above, in the conventional method of cultivating a cell ortissue of a living body, the cell is cultivated under a condition wherea temperature, a humidity, a concentration of carbon dioxide and aconcentration of oxygen are optimally set in an incubator. In such aculture in the incubator, it is a two-dimensional culture on alaboratory dish, and a three-dimensional culture has been now tried.Further, in such a culture method, a culture medium, cell or tissue thatis exposed by an open air is prone to contamination by bacteria, so thatstable culture is hardly performed.

Further, since the cell of a living body is always placed under physicalstimulation and the stimulation indirectly affects the control ofmetabolism of a cell, a cell division cycle, a concentration gradientand dispersion of living body stimulation, and hence stable culture ishardly realized, and further, it has been hard to set or change theamount, variation, cycle of the physical stimulation. More stillfurther, delicate set and regulation of a pressure are needed inculture, which requires a skill of a person in charge of culture.

Accordingly, in the conventional in vitro culture of a living cell, ittakes time for a cell to grow to the same size as a cell to be restored,and hence there occurs a case where a normal culture is marred bycontamination.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a method of and anapparatus for cultivating a cell or tissue capable of preventing thecell or tissue from being contaminated, and of realizing an efficient invitro culture.

According to the invention, a culture position (culture chamber) isinstalled under an arbitrarily controlled environment such as anenvironment mimicking the living body, a culture medium is suppliedwhile a cell or tissue is held in the foregoing culture position so thatthe cell or tissue is cultivated at the culture position under an idealenvironment, whereby the cell or tissue is prevented from contaminationand an efficient in vitro culture thereof can be realized.

To achieve the above object, the method of cultivating a cell or tissueaccording to the first aspect of the invention is characterized incomprising holding a cell or tissue of a living body at a specificculture position, setting the cell or tissue under an environmentmimicking the living body, supplying a culture medium to the cell ortissue, and cultivating the cell or tissue at the culture position.

That is, it is ideal that a tissue necessary for restoring a damagedtissue of a living body and so forth is used by a cell or tissue of thesame living body. It is necessary to cultivate to perform an in vitroculture using a cell or tissue collected from the living body to realizeit. It is important for the in vitro culture that the contamination isprevented and a culture environment likewise a living body, namely, anenvironment mimicking the living body is artificially realized. For thisend, a culture position is set under an environment formed artificiallyand the cell or tissue is held at a culture position and the culturemedium is supplied to realize the in vitro culture of the cell ortissue. An environment means living condition including internal orexternal stimulation needed for maintaining a life healthy on the basisof a living body formed of a cell or tissue. The culture medium includesa nutrition source needed for maintaining the life of a cell or tissueand growing it. In this case, the supply of the culture medium applies ahydraulic pressure and physical stimulation to the cell or tissue, sothat the cell or tissue is susceptible to metabolism functions, celldivision cycle, concentration gradient or dispersion of living bodystimulation so that the culture is enhanced. As a result, it is possibleto cultivate the cell or tissue which is close to a tissue in a livingbody and easily fusible with a tissue in a living body.

The method of cultivating a cell or tissue according to the secondaspect of the invention is characterized in comprising holding a cell ortissue of a living body at a specific culture position (culturechamber), setting the cell or tissue under an environment mimicking theliving body, supplying continuously or intermittently a culture mediumto the cell or tissue by way of a culture circuit (culture circuit),applying a pressure which is varied continuously, a pressure which isvaried intermittently or a pressure which is varied periodically to thecell or tissue, and cultivating the cell or tissue at the cultureposition.

The setting of the culture position and environment are the same asmentioned above. The culture medium is continuously or intermittentlysupplied to the cell or tissue that is set at the culture positionthrough the culture circuit. When the culture medium is supplied to thecell or tissue through the culture circuit that is separated orintercepted from the outside, the mode of supply of the culture mediumcan be made continuous or intermittent, and also the prevention ofcontamination can be achieved. It is possible to mimic a living body andto cultivate the cell or tissue efficiently by controlling the mode ofthe supply of the culture medium, corresponding to a living bodyenvironment. A desired pressure acts on the cell or tissue under culturethereof to apply physical stimulation. The mode of application ofpressure is varied continuously, intermittently or periodically to mimica living body, and apply a physical or mechanical strength needed for aliving body such as flexibility or durability needed for the cell ortissue to be cultivated. Accordingly, it is possible to contribute tothe culture of a cell or tissue which is ideal or practical,corresponding to a living body at a specific position of a living body,namely, contribute to cultivate the cell or tissue which is close to atissue in a living body and easily fusible with a tissue in a livingbody.

The method of cultivating a cell or tissue according to the third aspectof the invention is characterized in further providing holding means forholding the cell or tissue to be cultivated at the culture position in asuspending or non-suspending state in the culture medium. That is, anexperiment confirmed that the cell or tissue to be cultivated is held ina static state, that is needed for enhancing a culture efficiency.

The method of cultivating a cell or tissue according to the fourthaspect of the invention is characterized in that the holding meansemploys a hydro-gel for holding the cell or tissue to be cultivated atthe culture position in a suspending state in the culture medium or ascaffold for holding the cell or tissue and absorbing the cell or tissuewhen it grows. That is, the cell or tissue to be cultivated may be heldin any way, and hydro-gel or scaffold is one example of holding the cellor tissue in this case. The hydro-gel is means for wrapping and holdingthe cell or tissue to be cultivated in a suspending state, and the cellor tissue can be taken out from the hydro-gel when the culture iscompleted. Further, the scaffold may comprise a porous body formed ofprotein, and the cell or tissue to be cultivated is held by the scaffoldand absorbs the scaffold as a nutrition as it grows.

The method of cultivating a cell or tissue according to the fifth aspectof the invention is characterized in that the culture medium includesone or not less than two of amino acids of various types, saccharides,salts and protein. That is, it is possible to use the culture mediumcorresponding to the cell or tissue to be cultivated, for example, it ispossible to use one of amino acids of various types, saccharides, saltsand protein or not less than two of materials selected therefrom or allof these materials. The selection of the culture medium is an essentialelement for efficient culture or for forming the cell or tissue withhigh quality.

The method of cultivating a cell or tissue according to the sixth aspectof the invention is characterized in that the environment mimicking theliving body under which the cell or tissue is cultivated is setdepending on physiological conditions of the living body at a specificposition, an age, a height, a weight, a sex of the living body and otherinformation inherent in the living body in addition to the physiologicalconditions of the living body. That is, it is very important that thecell or tissue for use in restoring a part of a living body conformed tothe living body, and the culture environment can be set by use ofinformation inherent to the living body serving as one element.

The method of cultivating a cell or tissue according to the seventhaspect of the invention is characterized in that the environmentmimicking the living body under which the cell or tissue is cultivatedis set by a nitrogen gas, an oxygen gas, a carbon dioxide gasrespectively supplied through the culture medium, a temperature and ahumidity. That is, since it is desirable that an environment under whichthe cell or tissue is cultivated corresponds to a living body, forexample, if nitrogen gas, oxygen gas or carbon dioxide gas is suppliedto a cultivation space and a temperature or humidity is set to thatadapted for cultivation, a living body environment can be controlled toa desired state.

The method of cultivating a cell or tissue according to the eighthaspect of the invention is characterized in that the pressure applied tothe cell or tissue can be arbitrarily set depending on the specificposition of the living body. That is, it is possible to form an ideal orpractical cell or tissue by applying a pressure, corresponding to the aliving body at a specific position to be restored.

The method of cultivating a cell or tissue according to the ninth aspectof the invention is characterized in that the pressure applied to thecell or tissue is a pressure which is varied continuously, a pressurewhich is varied intermittently or a pressure which is variedperiodically or a pressure combining these pressures. That is, it ispossible to form the pressure pattern that is varied continuously,intermittently or periodically, and it is selected or combined torealize ideal physical stimulation so as to affect metabolism function,cell division cycle, concentration gradient or dispersion of living bodystimulation so that the culture is enhanced.

The apparatus for cultivating a cell or tissue according to the tenthaspect of the invention is characterized in comprising a culture unit(culture circuit unit) having a culture chamber containing therein acell or tissue and supplying culture medium, pressure application means(pressure application apparatus) for applying a pressure to the cell ortissue in the culture chamber, and culture medium supply means (culturemedium supply apparatus) for intermittently or continuously supplyingthe culture medium to the culture unit.

That is, the culture unit accommodates the cell or tissue to becultivated in the culture chamber to supply a culture medium needed forthe cell or tissue that is intercepted from the open air. The cell ortissue that is intercepted from the open air is protected fromcontamination by bacteria and so forth, and hence it grows to a tissuehaving an excellent quality. A desired pressure by pressure applicationmeans in addition to physical stimulation caused by a hydraulic pressureand a flow by the culture medium is applied to the cell or tissue. As aresult, it affects metabolism function, cell division cycle,concentration gradient or dispersion of living body stimulation so thatthe culture is enhanced. The mode of supply of the culture medium to thecell or tissue is arbitrarily set by the culture medium supply means,and the culture medium can be supplied to the cell or tissueintermittently or continuously so that the culture is enhanced by avariety of physical stimulation. The mode of the supply of the culturemedium includes one of or both of the supply of a new culture medium atall times or the supply of the culture medium by repetitivelycirculating the culture medium. In the mode of circulation of theculture medium can save the culture medium, but there is an advantage ofthe prevention of the variation in concentration of the culture mediumwhen supplying the culture medium in one direction.

The apparatus for cultivating a cell or tissue according to the eleventhaspect of the invention is characterized in further providing controlmeans for controlling the pressure application means or culture mediumsupply means. That is, although the pressure application means orculture medium supply means can be controlled arbitrarily, variouscontrols such as a feed back control or feed forward control and aprogram control and so forth can be performed by use of control meanssuch as a computer. It is needless to say to add a personal collectioncontrol by an interruption, and the collection control is not excluded.

The apparatus for cultivating a cell or tissue according to the twelfthaspect of the invention is characterized in that the pressure appliedfrom the pressure application means to the cell or tissue can bearbitrarily set depending on the cell or tissue. The manner of applyinga pressure, namely, a pressure pattern is set, corresponding to a cellor tissue to be cultivated, thereby performing an efficient culture.

The apparatus for cultivating a cell or tissue according to thethirteenth aspect of the invention is characterized in that the pressureapplied from the pressure application means to the cell or tissue is apressure which is varied intermittently, a pressure which is repeatedevery given time or a pressure which increases or decreases every giventime. That is, the pressure pattern can be conceived in all modes,thereby cultivating cell or tissue efficiently by selecting a mode ofpressure pattern.

The apparatus for cultivating a cell or tissue according to thefourteenth aspect of the invention is characterized in that the cultureunit is independent of and detached from a culture apparatus body. Thatis, the culture unit having the culture chamber for accommodatingtherein the cultivated cell or tissue can be independent of and detachedfrom a culture apparatus body so that the cell or tissue can be movedtogether with the culture unit that is separated from the open air toprotect the cell or tissue from being contaminated by bacteria duringthe motion thereof.

The apparatus for cultivating a cell or tissue according to thefifteenth aspect of the invention is characterized in that the cultureunit is accommodated in a hermetically sealed space that is interceptedfrom an open air. That is, since the hermetically sealed space is theculture space, and it is intercepted from an open air, it is possible toset a culture environment by the supply of the desired gas, to protectthe cell or tissue from the contamination by the open air.

The apparatus for cultivating a cell or tissue according to thesixteenth aspect of the invention is characterized in that the cultureapparatus further comprises gas absorption means capable of absorbing anitrogen gas, an oxygen gas, a carbon dioxide gas. That is, a nitrogengas, an oxygen gas, a carbon dioxide gas can be supplied to the cultureunit accommodated in the hermetically sealed space and the gasabsorption means is provided in the culture unit so that the gas isapplied to the cell or tissue and a living environment can be mimickedby supplying and controlling gas.

The apparatus for cultivating a cell or tissue according to theseventeenth aspect of the invention is characterized in that thehermetically sealed space is filled with a nitrogen gas, an oxygen gas,a carbon dioxide gas. That is, when a nitrogen gas, an oxygen gas, acarbon dioxide gas is filled in the culture space formed by thehermetically sealed space, a living body environment can be mimicked.

The apparatus for cultivating a cell or tissue according to theeighteenth aspect of the invention is characterized in furthercomprising a culture medium tank for storing therein the culture mediumto be supplied to the culture unit. That is, the culture medium supplysource is needed for supplying or circulating a necessary culture mediumto the culture unit, and the culture medium tank is a supply source.Particularly, it is possible to prevent the culture medium held in theculture unit from being contaminated, if the culture medium tank isinstalled in the hermetically sealed space that is intercepted from theopen air.

The apparatus for cultivating a cell or tissue according to thenineteenth aspect of the invention is characterized in that the culturechamber includes a pressure transmitting film for receiving a pressurefrom the outside. That is, it is possible to apply pressure applicationstimulation to the cell or tissue accommodated in the culture chamber ina state wherein it is intercepted from an open air, and to realizedesired pressure application stimulation such as stimulation mimicking aliving body environment by providing the pressure transmitting film.

The apparatus for cultivating a cell or tissue according to thetwentieth aspect of the invention is characterized in that the culturechamber includes pressure buffering means. That is, it is possible torealize physical stimulation close to a living body environment and toenhance the culture of the cell or tissue by regulating a pressure bypressure buffering means when a part of a culture unit is pressurized.

The apparatus for cultivating a cell or tissue according to thetwenty-first aspect of the invention is characterized in that theapparatus in the tenth aspect of the invention further comprises apressure chamber fixed to the culture chamber by way of a pressuretransmitting film, and a pressure is applied to the cell or tissue inthe culture chamber by allowing a hydraulic pressure, an oil pressure oran air pressure to act on the cell or tissue in the culture chamber.That is, it is possible to realize desired pressure applicationstimulation and to mimic a living body environment with high accuracy byusing any of the hydraulic pressure, the oil pressure or the airpressure as pressure forming means.

The apparatus for cultivating a cell or tissue according to thetwenty-second aspect of the invention is characterized in that theculture medium supply means comprises a medium supply chamber providedin the culture unit and a medium supply unit for pressuring a culturemedium that is taken in the medium supply chamber and supplying thepressurized culture medium. That is, the culture medium supply means ismeans for supplying and circulating the culture medium in the cultureunit, and it is formed of various types, for example, if it is formed ofthe medium chamber and the medium supply unit for pressuring a culturemedium that is taken in the medium supply chamber, the amount of appliedpressure can be controlled to set a desired amount of supply medium.

The apparatus for cultivating a cell or tissue according to thetwenty-third aspect of the invention is characterized in that a reliefvalve is provided in the culture, and when a pressure of the culturemedium exceeds a given pressure which is arbitrarily set to the reliefvalve, the relief valve is opened to decrease the pressure of theculture medium. That is, it is important to buffer the pressure to beapplied to the culture for applying ideal pressure applicationstimulation to the cell or tissue. If the pressure relieve valve is usedas one means, and it is opened to decrease the pressure of the culturemedium when the pressure of the culture medium exceeds a given pressurewhich is arbitrarily set to the relief valve, the culture medium iscontrolled in an ideal pressure state without contaminating the culturemedium.

The apparatus for cultivating a cell or tissue according to thetwenty-fourth aspect of the invention is characterized in that heatingmeans or humidifying means are provided in a hermetically sealed spaceand the hermetically sealed space is kept and controlled at a desiredtemperature or humidity. That is, it is possible to provide a culturespace conforming to a living body environment by controlling atemperature and a humidity of the hermetically sealed space in which theculture unit is accommodated.

The apparatus for cultivating a cell or tissue according to thetwenty-fifth aspect of the invention is characterized in that a soundproducing unit for applying a super-sound wave or the like sound wave inthe culture chamber in the culture unit. That is, it is possible tomimic a living body environment acoustically by using the soundproducing unit together because a living body receives acousticstimulation from the outside, and possible to inject the cell or tissueto be cultivated in a culture chamber by use of a super-sound wavetogether with high reliability.

The apparatus for cultivating a cell or tissue according to thetwenty-sixth aspect of the invention is characterized in that theapparatus further comprises control means for controlling concentrationof a gas to be supplied to the hermetically sealed space. That is, it ispossible to mimic a living body environment to enhance the culture ofthe cell or tissue by controlling the concentration of a gas to besupplied to the hermetically sealed space by controlling means.

The objects, features and advantages of the invention are now made moreclear with reference to the following first to fourth embodiments of theinvention, detail descriptions thereof and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing a method of and an apparatus forcultivating a cell or tissue according to a first embodiment of theinvention;

FIG. 2 is a view showing the method of and the apparatus for cultivatinga cell or tissue in FIG. 1.

FIG. 3 is an enlarged view of a part of a culture circuit unit, aculture medium supply apparatus, a pressure application apparatus, and apressure buffering apparatus respectively of the culture apparatus;

FIG. 4 is a view showing a status where the culture apparatus and theculture circuit unit are separated from each other;

FIG. 5 is a block diagram showing a control apparatus;

FIG. 6 is a flow chart showing a method of cultivating the cell ortissue according to the invention;

FIG. 7 is a flow chart showing initialization in the method ofcultivating the cell or tissue according to the invention;

FIG. 8 is a flow chart continued from FIG. 7 showing initialization inthe method of cultivating the cell or tissue according to the invention;

FIG. 9 is a flow chart continued from FIG. 8 showing initialization inthe method of cultivating the cell or tissue according to the invention;

FIG. 10 is a view showing a displacement of a pressure applicationpiston in the pressure application apparatus and a pressure chamberrelative to the motion of the pressure application piston;

FIG. 11 is a view showing a pressure of a adjustable valve relative to adisplacement of an actuator in a pressure relief valve;

FIG. 12 is a timing chart showing the execution a culture mode at avariable pressure;

FIG. 13 is a timing chart showing another execution of a culture mode atthe variable pressure;

FIG. 14 is a front view of a culture apparatus in a method of and anapparatus for cultivating a cell or tissue according to a secondembodiment of the invention;

FIG. 15 is a side view of the culture apparatus unit in FIG. 14;

FIG. 16 is a view showing a part of a culture apparatus body and aculture circuit unit in FIG. 14;

FIG. 17 is a view of the culture circuit unit separated from the cultureapparatus body in FIG. 16;

FIG. 18 is a partial sectional view showing a part of the cultureapparatus body from which the culture circuit unit in FIG. 16is removed;

FIG. 19 is a partial sectional view showing a pressure applicationapparatus of the culture circuit unit in FIG. 16;

FIG. 20 is a partial sectional view of a culture medium supply apparatusin FIG. 14;

FIG. 21 is a partially sectional view of a pressure buffering apparatusin FIG. 14;

FIG. 22 is a partially sectional view of a culture medium supplyapparatus according to a modification of the second embodiment of theinvention.

FIG. 23 is a view showing a method of and an apparatus for cultivating acell or tissue according to a third embodiment of the invention;

FIG. 24 is a view showing a method of and an apparatus for cultivating acell or tissue according to a fourth embodiment of the invention;

FIG. 25 is a view showing a pressure application operation or controlaccording to a fourth embodiment of the invention;

FIG. 26 is a view showing a conventional method of and an apparatus forcultivating a cell or tissue; and

FIG. 27 is a view showing another conventional method of and anapparatus for cultivating a cell or tissue.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

First Embodiment (FIGS. 1 to 13)

A method of and apparatus for cultivating a cell or tissue according toa first embodiment of the invention is described with reference to FIGS.1 to 13.

FIG. 1 is a block diagram showing the method of and th apparatus forcultivating a cell or tissue according to the first embodiment of theinvention;

A culture apparatus 1 for realizing the method of cultivating a cell ortissue has a hermetically sealed space 2 as a culture space in which aculture circuit unit 4 serving as a culture unit to supply culturemedium 3 to cell or tissue to be cultivated is installed.

The culture circuit unit 4 can be set up so as to be separated ordetachable from a body of the culture apparatus 1 (hereinafter referredto as culture apparatus body). The culture circuit unit 4 includes aculture medium tank 9, culture medium supply apparatus 6, a culturepressure application apparatus 8, a gas absorption apparatus 10, a valve11, and a branched path 13 having a valve 15 thereon. The culture medium3 is a carrier for supplying a nutrition to the cell or tissue to becultivated and a fluid including essential amino acid and various aminoacids, glucose (saccharide), and an sometimes inorganic material such asNa⁺, Ca⁺⁺ is added thereto depending on the cell or tissue to becultivated or a protein such as serum is included therein. Further,these apparatus are formed of a resin material having a sufficient heatresistance and does not melt to produce a material that affects a livingbody such as a fluorine resin, PEEK, a high grade heat resistantpolypropylene, silicone or stainless steel, thereby preventing theconstituents from being contaminated.

The valves 11, 15 may be formed of a pinch valve and so forth. Theculture circuit unit 4 forms a closed loop circuit when the valve 15 isshut and the valve 11 is opened, an entire open loop circuit when thevalve 15 is opened and the valve 11 is shut, and a partial open loopcircuit when both the valves 11, 15 are opened. The culture circuit unit4 may includes a gas absorption portion 41 denoted by two dotted onechain line and a pressure resistant portion 43 denoted by a solid lineinstead of the gas absorption apparatus 10 that is partially installedtherein. The gas absorption portion 41 is a portion to render gas filledin the hermetically sealed space 2 to be absorbed by the culture medium3 while the pressure resistant portion 43 is a portion to assure areliable medium supply, corresponding to the pressure applicationportion of the culture medium 3 so as to prevent leakage of medium. Atube formed of an elastomer material through which gas easily passes agas such as CO₂, O₂ may be used in the gas absorption portion 41.

The culture medium tank 9 is accommodated in the hermetically sealedspace 2 and means for storing therein the culture medium 3 that isneeded for cultivating the cell or tissue. The culture medium supplyapparatus 6 is means for supplying the culture medium 3 to the culturecircuit unit 4, namely, when a medium supply apparatus 12 that isinserted into the culture circuit unit 4 is driven by a drivingapparatus 14, it supplies a predetermined amount of culture medium 3 tothe culture circuit unit 4. The culture pressure application apparatus 8is means for applying a pressure to a cell 5 (FIG. 3) or tissue to becultivated, and includes a pressure application apparatus 16 and apressure buffering apparatus 18. The pressure application apparatus 16comprises a culture chamber 20 of the culture circuit unit 4, a pressurevessel 22 attached to the culture chamber 20 and a driving apparatus 24for allowing an arbitrary pressure to act on the culture chamber 20. Acell or tissue to be cultivated is transplanted in a scaffold formed ofa collagen and so forth and it is accommodated in the culture chamber 20and is separated from the outside.

The pressure buffering apparatus 18 is means for buffering a pressure tobe applied to the culture medium 3 by the culture pressure applicationapparatus 8, and it sets a pressure of the culture medium 3 exceeding apredetermined value as the maximum pressure by driving a pressure reliefvalve 26 that is inserted into the culture circuit unit 4 by a drivingapparatus 28. When a pressure of the culture medium 3 exceeding themaximum pressure acts on the culture circuit unit 4, the pressurebuffering apparatus pressure 18 operates the pressure relief valve 26 toallow the culture medium 3 to escape therefrom, thereby buffering thepressure. A pressure application fluid is introduced into the pressurevessel 22 from a pressure application fluid introduction apparatus 30provided together with the culture pressure application apparatus 8.

A humidity regulating apparatus 32, a temperature regulating apparatus34, and a gas mixture/concentration regulating apparatus 36 areinstalled in the culture apparatus 1 to regulate an atmospherichumidity, an atmospheric temperature and gas mixture and concentration.An operation apparatus 38 and a control apparatus 40 are respectivelyinstalled in the culture apparatus 1, wherein desired control operationsare performed by an administrator using the operation apparatus 38 whilethe control apparatus 40 is means for controlling a various apparatussuch as the culture medium supply apparatus 6, culture pressureapplication apparatus 8, pressure application fluid introductionapparatus 30, humidity regulating apparatus 32, temperature regulatingapparatus 34, gas mixture/concentration regulating apparatus 36 inresponse to an operation input or a control program through theoperation apparatus 38.

The method of cultivating the cell or tissue using the culture apparatusis described next. Indispensable items such as culture conditions areinputted to the control apparatus 40 by operating the operationapparatus 38 for preparing culture.

In this case, the indispensable items are various pressures set in theculture medium 3, and they are set to a mode of, for example, a maximumpressure, a minimum pressure, a pressure gradient such as increase ordecrease of pressure, a pressure application period, the amount of flowof the culture medium 3, a culture temperature and culture time. Theculture circuit unit 4 selectively switches between the valves 11, 15 torender them to open or shut so as to form a closed loop or an open loop.

Then a scaffold 7 (FIG. 3) formed of a sponge such as collagen isprovided in the culture chamber 20 and the cell 5 (FIG. 3) or tissue tobe cultivated is transplanted in the scaffold 7. The sponge such ascollagen may be formed by freezing or drying collagen fluid inside theculture chamber 20.

Subsequently, a prescribed amount of culture medium 3 is introduced intothe culture medium tank 9, and the hermetically sealed space 2 isclosed, then the operation switch is turned on so as to prepare theculture operation (automatic operation) so that a pressure applicationfluid is supplied from the pressure application fluid introductionapparatus 30 to the pressure vessel 22 side.

When the culture medium supply apparatus 6 is driven, the culture medium3 flows to the culture chamber 20 side through the medium supplyapparatus 12 so that the culture medium 3 is supplied to the cell ortissue to be cultivated. The mode of supplying the culture medium 3 isselected from a continuous supply, an intermittent supply, a periodicsupply or the combination thereof.

The cell or tissue held by the scaffold is accommodated in the culturechamber 20 filled with the culture medium 3 and a pressure is appliedfrom the pressure vessel 22 to the cell or tissue. The mode of pressuredepends on pressure patterns set during the preparation of culture.

When a pressure applied to the culture medium 3 exceeds a set pressure,the culture medium 3 flows out from the pressure resistant portion 43through the pressure relief valve 26 so that a pressure is controlled.

If such operations are repeated during a prescribed culture time, thecell or tissue grows until it reaches a desired size inside the culturechamber 20. If a sponge such as collagen is used as a scaffold, the cellor tissue to be cultivated absorbs the collagen so that the scaffolddisappears naturally.

If a hydro-gel is used as holding means, the cell or tissue isaccommodated and held inside the hydro-gel in a suspending state.

If the culture circuit unit 4 forms the closed loop when the valve 15 isshut and the valve 11 is opened, the culture medium 3 circulates insidethe culture circuit unit 4 and it is supplied to the cell or tissue sideto be cultivated. If the culture circuit unit 4 forms the opened loopwhen the valve 11 is shut and the valve 15 is opened, the culture medium3 flows to the branched path 13 side then to the pressure applicationfluid introduction apparatus railroad ties 30 side, namely, to a watertank for pressurizing 68 side (FIG. 2) so that fresh culture medium 3can be always supplied to the cell or tissue side to be cultivated.

A gas such as nitrogen, oxygen, carbon dioxide is absorbed by the gasabsorption apparatus 10 or gas absorption portion 41 of the culturecircuit unit 4 from the interior of the hermetically sealed space 2during the culture and supplied to the culture medium 3 so that a gasthat is needed for gas exchange like a living body is supplied to thecell or tissue through the culture medium 3.

In such a manner, a culture environment mimicking the living body is setin the cell or tissue so that an in vitro culture can be efficientlyperformed while it is not contaminated by bacteria and so forth. Thatis, since a hydraulic pressure of the culture medium 3 and physicalstimulation by the flow of the culture medium 3 are applied to the cellor tissue inside the culture chamber 20, the cell or tissue issusceptible to metabolism function, cell division cycle, concentrationgradient or dispersion of living body stimulation so that the culture isenhanced. Further, the cell or tissue is subjected to physicalstimulation by a pressure application by the pressure applicationapparatus 16 or depending on the mode of pressure application.Accordingly, the culture of the cell or tissue is enhanced so that atissue close to that in the living body or a tissue easily fuses withthe tissue of the living body can be cultivated. Still furthr, if thepressure resistant portion 43 is partially provided, a cost needed forthe pressure resistant construction can be reduced.

FIG. 2 shows a detailed construction of the culture apparatus 1 and FIG.3 enlarges a part of the culture circuit unit 4, culture medium supplyapparatus 6, pressure application apparatus 16 and pressure bufferingapparatus 18 of the culture pressure application apparatus 8respectively installed in the culture apparatus 1. The culture apparatus1 is constructed, as shown in FIG. 4, such that the culture circuit unit4 is detachable from the culture apparatus 1.

The culture apparatus 1 has a culture box 42 that can be hermeticallysealed, and the opening and shutting of a door 270 (FIG. 14) is detectedby a door switch 44. The culture circuit unit 4 for supplying theculture medium 3 is accommodated in the culture box 42. The culturecircuit unit 4 is a detachable tube unit for connecting a culture mediumbag 48 serving as a culture medium tank for storing therein the culturemedium 3 by way of the culture chamber 20, medium supply apparatus 12,and pressure relief valve 26 by tubes 50A, 50B, 50C, 50D and 50E. Thetubes 50A, 50D, 50E constituting gas absorption portion 41 (FIG. 1) areformed of a vent tube made of an elastomer material or the like capableof absorbing gas inside the culture box 42. The tubes 50B and 50Cconstituting the pressure resistant portion 43 (FIG. 1) are formed of apressure resistant tube capable of withstanding a pressure of theculture medium 3. A gas absorption portion 52 for absorbing the gasinside the culture circuit unit 4 is formed in the tube 50E by bendingthe tube 50E.

The culture medium bag 48 is supported by a hook 56 having a detectionswitch 54 serving as weight detection means on the wall surface of theculture box 42, wherein a volume corresponding to the weight of theculture medium 3 inside the culture medium bag 48 is detected by thedetection switch 54. When the detection switch 54 detects the decreaseof a predetermined weight of the culture medium bag 48, abnormality isnotified by indication means (indication apparatus 232) or telephone orthe like by way of the control apparatus 40. A culture medium dischargeportion 58 is provided at the branched portion between the tubes 50A and50E where the medium supply apparatus 12 and gas absorption portion 52are provided, and it is opened or shut by a checking valve 59. Thechecking valve 59 is means for collecting the culture medium 3 insidethe culture circuit unit 4, and the culture medium 3 collected throughthe culture medium discharge portion 58 is subjected to an inspection ofdenaturation, namely, it is inspected whether the culture medium 3 iscontaminated by bacteria or the like or subjected to an inspection ofpH, concentration, material produced by the culture medium 3, oxygenconcentration, carbon dioxide concentration and so forth.

The cell 5 to be cultivated is transplanted in the scaffold 7 formed bycollagen or the like and it is accommodated inside the culture chamber20 together with the scaffold 7. The culture chamber 20 is formed of aculture vessel 61 that is detachably attached to a pressure chamber 60by fixing means such as a plurality of bolts 62 or the like, and aninjection port 63 is provided onto the culture vessel 61. The injectionport 63 is used for transplanting the cell 5 to be cultivated in thescaffold 7 provided inside the culture chamber 20 by a syringe or thelike. The culture chamber 20 can be attached to the pressure chamber 60by other fixing means such as a clamper. Both the pressure chamber 60and culture vessel 61 are sealed by a seal member such as an O ring. Thesurface of the pressure chamber 60 side of the culture chamber 20 isclosed by a pressure transmitting film 64 to form a hermetically sealedspace and water 65 (for pressurizing) inside the pressure chamber 60contacts the culture chamber 20 by way of the pressure transmitting film64.

The water (fluid) tank for pressurizing 68 is connected to the pressurechamber 60 through a water supply conduit 66, and a flowing water sensor70, a pump 80, a bypass valve 82, and a seal valve 84 are respectivelyprovided on the water supply conduit 66, wherein a bypass valve 82 isprovided on a bypass conduit 88 having an orifice 86 at the middlethereof. That is, when the bypass valve 82 and seal valve 84 are openedto drive the pump 80, when the water 65 (for pressurizing) is filledinside the pressure chamber 60 from the water tank for pressurizing 68.Since a level of pressurized water inside the water tank forpressurizing 68 is detected by a water level sensor 96, when a watersupply valve 92 is opened or shut depending on a water level, the water65 (for pressurizing) can be replenished in the water tank forpressurizing 68 through a water supply conduit 94 so that the waterlevel inside the water tank for pressurizing 68 is always kept in theoptimum water level. A water discharge conduit 98 is branched from thewater supply conduit 66 of the water tank for pressurizing 68, and thewater 65 (for pressurizing) is discharged through the water dischargeconduit 98 when a fluid discharge valve 100 is opened when the cultureof the cell 5 is completed.

A collection conduit 102 directing toward the water tank forpressurizing 68 is provided in the pressure chamber 60 and there areprovided a seal valve 104 and a circulation pump 106 in the collectionconduit 102. The tip end of the collection conduit 102 is submerged inthe water 65 (for pressurizing) inside the water tank for pressurizing68. That is, when the seal valve 84 is opened and the bypass valve 82 isshut to drive the circulation pump 106, a pressure inside the pressurechamber 60 is decreased, air bubbles and so fourth that are stuck toinner walls of the pressure chamber 60 and the water supply conduit 66,collection conduit 102 and so forth can be discharged toward the watertank for pressurizing 68 side. Further, the water 65 (for pressurizing)inside the pressure chamber 60 can be supplied from the water tank forpressurizing 68 to the pressure chamber 60 through the water supplyconduit 66 when the pump 80, circulation pump 106 are simultaneouslydriven while it can be returned to the water tank for pressurizing 68through the collection conduit 102 so that it can be circulated betweenthe pressure chamber 60 and the water tank for pressurizing 68.

A heater 108, a temperature sensor 110, a pressure sensor 112 and asound producing unit 114 are respectively provided on the wall surfaceof the pressure chamber 60, wherein heating, temperature and pressure ofthe water 65 (for pressurizing) accommodated inside the pressure chamber60 are detected by the temperature sensor 110, pressure sensor 112, andwherein a sound wave such as an ultrasonic wave from the sound producingunit 114 can be applied to the pressure chamber 60, if need be.

A pressure application piston 116 as pressure application means isreciprocatively provided inside the pressure chamber 60 wherein thepressure application piston 116 is supported by a supporter cylinder 117protruded from the wall surface of the pressure chamber 60, and O-ring119 serving as seal means seals between the supporter cylinder 117 andpressure application piston 116. An actuator 120 serving as pressureapplication driving means and a motor 122 are fixed to the pressureapplication piston 116 by way of a pressure application spring 118. Themotor 122 is formed of, e.g., a stepping motor, and the rotation of themotor 122 is converted into a reciprocating motion by the actuator 120and the reciprocating motion is applied to the pressure applicationspring 118 whereby a pressure inside the pressure chamber 60 can beincreased or decreased depending on the reciprocating motion of thepressure application piston 116 so that a high pressure is produced whenthe pressure application piston 116 is moved forward while a lowpressure is produced when the pressure application piston 116 is movedbackward, and the variation in pressure applies pressure applicationstimulation to the cell 5 in the scaffold 7 through the pressuretransmitting film 64. Further, the position of the pressure applicationpiston 116 is detected by a position sensor 123, and the detected datais used for controlling the reciprocating motion of the pressureapplication piston 116, namely, the control of pressure applicationstimulation.

In this case, the water 65 (for pressurizing) is filled in the pressurechamber 60, and a pressure applied by the pressure application piston116 acts on the entire surface of the pressure transmitting film 64through the water 65 (for pressurizing), and the same pressure servingas a hydrulic pressure uniformly acts on the cell 5 or tissue throughthe pressure transmitting film 64 and the culture medium 3, and a straincaused by the same pressure can also act on the cell 5 or tissue.Further, it is possible to allow a dynamic range of the amount ofvariation in pressure large when controlling the amount of motion of thepressure application piston 116 so as to finely control the pressurefrom a small value to a large value. The motion of the pressureapplication piston 116 is detected by the position sensor 123 and ismonitored by the control apparatus 40, and when the amount of motionarrives a critical position, an alarm output is outputted from thecontrol apparatus 40 as an abnormality of the culture apparatus 1, thenalarm indication is performed on indication means (the indicationapparatus 232 in FIG. 5 and so forth) connected to the control apparatus40 or the abnormality is notified to an administrator through acommunication line such as a telephone.

The medium supply apparatus 12 for supplying continuously orintermittently the culture medium to culture chamber 20 includes amedium supply chamber 128 having a supply side check valve 124 and asuction side check valve 126 at the medium input and output side, and itis detachably attached to the culture box 42 by screws 130. A mediumsupply piston 132 is reciprocatively attached to the medium supplychamber 128, and a disinfectant fluid tank 134 is provided at the middleportion of the medium supply piston 132, while a pressure applicationspring 136 is attached to the middle portion of the medium supply piston132. O-rings 133, 135 serving as seal means are provided between themedium supply piston 132 and a main body of the medium supply chamber128. A disinfectant or an antiseptic solution or antibiotic substancesuch as penicillin is filled in the disinfectant fluid tank 134 toprevent the entrance of bacteria or a foreign matter from the outside.The pressure application spring 136 is accommodated in a protectioncylinder 137.

An actuator 138 serving as driving means and a motor 140 arerespectively attached to the rear end portion of the medium supplypiston 132. The motor 140 is formed of, e.g., a stepping motor and therotation of the motor 140 is converted into a reciprocating motion bythe actuator 138, and the thus converted reciprocating motion is appliedto the pressure application spring 136 so that a pressure inside themedium supply chamber 128 increases or decreases in response to thereciprocating motion of the medium supply piston 132. The variedpressure at that time is applied to valve bodies 142, 144 of the supplyside check valves 124 and suction side check valve 126. When the mediumsupply piston 132 is pulled out from the medium supply chamber 128, theinside of the medium supply chamber 128 is negatively pressurized by theamount of pulling out of the medium supply piston 132 so that the valvebody 142 is pulled downward by a restoring force of a spring 143 to shutthe supply side check valve 124 while the valve body 144 is pulledupward against a pressure application force of a spring 145 to open thesuction side check valve 126. As a result, the culture medium 3 issucked into the medium supply chamber 128. Further, when the mediumsupply piston 132 moves inside the medium supply chamber 128, the insideof the medium supply chamber 128 is pressurized to lower the valve body144 so as to shut the suction side check valve 126 while the valve body142 moves upward to open the supply side check valve 124 so that theculture medium 3 inside the medium supply chamber 128 is supplied to theculture chamber 20 side.

The pressure buffering apparatus 18 for the culture medium 3 has thepressure relief valve 26, and the pressure relief valve 26 is detachablyattached to the culture box 42 by screws 146. A valve body 150 whichmoves back and forth and closable inside the valve chamber 148 isattached to the pressure relief valve 26, a disinfectant fluid tank 153is provided on the middle portion of a plunger 152 of the valve body150. O rings 155, 157 serving as seal means are provided between theplunger 152 and a main body of the valve chamber 148. A disinfectant oran antiseptic solution or an antibiotic substance such as penicillin isfilled in the disinfectant fluid tank 153 to prevent the entrance ofbacteria or a foreign matter from the outside. An actuator 156 servingas driving means and a motor 158 are respectively attached to the rearend portion of the plunger 152 of the valve body 150 by way of a bufferspring 154. The motor 158 is formed of, e.g., a stepping motor and therotation of the motor 158 is converted into a reciprocating motion bythe actuator 156, and the thus converted reciprocating motion is appliedto the buffer spring 154 so that an operation pressure for opening thevalve body 150 is controlled in accordance with the compression of thebuffer spring 154. That is, when the compression of the buffer spring154 is high, a pressure from the culture medium 3 needed for opening thevalve body 150 becomes high while when the compression of the bufferspring 154 is low, a pressure from the culture medium 3 needed foropening the valve body 150 becomes low. The reason why the pressurebuffering apparatus 18 is provided is to buffer a pressure applicationforce applied to the culture medium 3 in the culture chamber 20 at theculture circuit unit 4 side.

A pinch valve 162 and a suction tube 164 are branched from a tube 50Dfor connecting the valve chamber 148 of the pressure relief valve 26 andthe culture medium bag 48, and a pinch valve 166, a check valve 168 anda culture medium tank 170 are respectively provided on the suction tube164, while the culture medium tank 170 is connected to the collectionconduit 102 through a suction tube 165. The pinch valve 162 is used foropening and shutting the tube 50D while the pinch valve 166 is used foropening and closing the suction tube 164. A valve body 169 of the checkvalve 168 is shut by a pressure application force of a spring 171. Whena pressure of the culture medium 3 exceeds a pressure application forceof the spring 171, the culture medium 3 flows to the culture medium tank170 side through the suction tube 164. The pinch valve 166 can shut thesuction tube 164 by operating it regardless of the check valve 168 so asto prevent the flow of the culture medium 3. Since the culture mediumtank 170 serves as a hermetically sealed container when the pinch valve166 is opened, a pressure inside the culture medium tank 170 isdecreased when the circulation pump 106 is driven while closing the sealvalve 104 is shut so that the valve body 169 is moved against thepressure application force of the spring 171 to open the check valve168. At this time, the culture medium 3 can be drawn into the culturemedium tank 170 side.

An N₂ gas tank 172, an O₂ gas tank 174 and a CO₂ gas tank 176 serving asthe gas mixture/concentration regulating apparatus 36 are respectivelyconnected to the culture box 42 through conduits 178, 180, 182. Gasshutting valves 184, 186, 188, flow control valves 190, 192, 194, flowmeters 196, 198, 200, pressure control apparatus 202, 204, 206 andvalves 208, 210, 212 are respectively provided on the conduits 178, 180,182. That is, when the gas closing valves 184, 186, 188 are selectivelyopened or shut, at least one of N₂ gas, O₂ gas or CO₂ gas is suppliedand mixed with each other.

Humidifying water sourcer 216 for storing therein humidifying water 214serving as a humidity regulating apparatus 32 and a stirring fan 218 areinstalled in the culture box 42, while a gas heating heater 220 servingas a temperature regulating apparatus 34 as heating means, a boxtemperature sensor 222 and the stirring fan 218 are also installed inthe culture box 42. The stirring fan 218 is driven by a fan motor 224.

Although an alarm is issued when the culture apparatus 1 becomesabnormal, the control apparatus 40 controls a temperature, a gasconcentration inside the culture box 42 and continues a medium supplyoperation so as to hold the cell 5 or tissue under culture regardless ofthe kind of abnormality until an administrator takes necessary meansagainst the abnormality. The control apparatus 40 also controls atemperature, a gas concentration inside the culture box 42 and continuesa medium supply operation even if a predetermined culture time arrivesor a normal operation is completed.

FIG. 5 shows the concrete construction of the operation apparatus 38 andcontrol apparatus 40. A main control apparatus 230 is commonly providedin the operation apparatus 38 and control apparatus 40 formed of apersonal computer and so forth. An indication apparatus such as adisplay, liquid crystal, an external storage apparatus 234 such as ahard disc, an optical disc, a floppy disc, an IC card, and a key boardinput apparatus 236 are respectively connected to the main controlapparatus 230. The key board input apparatus 236 constitutes a part ofthe whole of the operation apparatus 38.

There are applied to the main control apparatus 230 the following,namely, a detection output of the temperature sensor 110 by way of atemperature detection circuit 238, a detection output of the boxtemperature sensor 222 by way of temperature detection circuit 240, adetection output of the pressure sensor 112 by way of a pressuredetection circuit 242, a detection output of the position sensor 123 anda detection output of the detection switch 54 while there are obtainedthe following respectively by way of the main control apparatus 230,namely, a driving output of the motor 122 by a driving circuit 244, adriving output of the motor 140 by a driving circuit 246, a drivingoutput of the motor 158 by a driving circuit 248, a driving output ofthe heater 108 by a driving circuit 250, a driving output of the valves184, 186, 188 by a driving circuit 252, a driving output of the fanmotor 224 by a driving circuit 254, a driving output of the heater 220by a driving circuit 256, and the driving output of the sound producingunit 114.

FIG. 6 is a flow chart showing a method of cultivating the cell ortissue according to the invention.

Step S1 is an initialization mode. This initialization mode includes astep of filling the water 65 (for pressurizing) inside the pressurechamber 60 and filling the culture medium 3 inside the culture circuitunit 4 after the culture circuit unit 4 is installed and a step ofsampling the amount of operation of the pressure application apparatus16 of the culture pressure application apparatus 8 and the pressurebuffering apparatus 18 corresponding to an inputted set pressure value,and storing the sampled amount of operation in a memory, describedlater. Elongation percentage of a material constituting the culturecircuit unit 4 is different from that of the pressure transmitting film64, and the amount of operation for obtaining a set pressure isdifferentiated by the presence of air valves and so forth remaining inthe pressure chamber 60. Accordingly, in the initialization mode, theseset values are corrected.

When the culture circuit unit 4 is installed, the gasmixture/concentration regulating apparatus 36, humidity regulatingapparatus 32 and temperature regulating apparatus 34 are operated,thereby filling gases inside the culture box 42 and regulating humidityand temperature to optimum values. Thereafter, the water 65 (forpressurizing) formed of service water and so forth is replenished in thewater tank for pressurizing 68 by opening the water supply valve 92,then the bypass valves 82, seal valves 84 and 104 are opened to operatethe pump 80 so that the water 65 (for pressurizing) is supplied insidethe pressure chamber 60. The amount of supply of the water 65 (forpressurizing) to the pressure chamber 60 is detected by the flowingwater sensor 70, and when a predetermined amount of water 65 (forpressurizing) is detected, the pump 80 is stopped so as to switch to acirculation operation by the circulation pump 106.

In the circulation operation, the bypass valve 82 is shut to switch tothe flow to the bypass conduit 88. At this time, the amount of water 65(for pressurizing) is restricted by the orifice 86 so that the pressurechamber 60 is negatively pressurized by the suction force of thecirculation pump 106, and air valves remaining inside the pressurechamber 60 are discharged toward the water tank for pressurizing 68side. At this time, the pinch valve 162 is shut and the pinch valve 166is opened so that the culture medium 3 inside the culture medium bag 48is filled in the culture chamber 20 by the negative pressure produced bythe circulation pump 106 through the tubes 50E, 50A, SOB. After theculture medium 3 is filled in the culture chamber 20 by operating thecirculation pump 106 for a predetermined time, the pinch valve 166 isshut and the pinch valve 162 and bypass valve 82 are opened to release anegative pressure caused by the circulated flow, then the circulationpump 106 is stopped. Subsequently, after the seal valves 84, 104 areshut, the water 65 (for pressurizing) inside the pressure chamber 60 isheated by the heater 108, and the heated temperature is detected by thetemperature sensor 110 to start the control of the temperature.

Then, the motor 158 of the pressure buffering apparatus 18 is operatedto shut the pressure relief valve 26 so as to block the tube 50C at agiven pressure. When the pressure application apparatus 16 is operatedby operating the motor 122 until a predetermined maximum pressure Pmaxis detected. When maximum pressure Pmax is detected, the counted numberof pulses of the motor 122 is stored in the memory of the main controlapparatus 230. Then, the motor 158 of the pressure buffering apparatus18 is rotated until the present pressure value is decreased, then thecounted number of pulses of the motor 158 is stored in the memory of themain control apparatus 230 while the pressure value serves as theposition of the maximum pressure Pmax.

Then, the motor 122 of the pressure application apparatus 16 is rotateduntil a predetermined minimum pressure Pmin is detected. When theminimum pressure Pmin is detected, the counted number of pulses of themotor 122 is stored in the memory of the main control apparatus 230.Subsequently, the motor 158 of the pressure buffering apparatus 18 isrotated, and the motor 158 is stopped at the position where the decreaseof a pressure from the minimum pressure Pmin starts. At this time, thecounted number of pulses of the motor 158 is stored in the memory of themain control apparatus 230.

Then, after the initialization mode, a program goes to step S2 todetermine whether it is a culture mode or not. That is, a pressure isperiodically varied to determine whether a culture is to be performed ornot, wherein when a pressure variation is to be controlled, the programgoes to a culture mode at varied pressure in step S3 while when theculture is performed at a given pressure, the program goes to a culturemode at fixed pressure in step S7.

In the culture mode at varied pressure in step S3, increasing of apressure, holding of a pressure, increasing of a pressure, and holdingof a pressure are repeated every cycle T to pressurize and stimulate thecell 5 in the culture chamber 20 while supplying the culture medium 3 tothe cell 5.

In step S4, it is determined whether each error between the pressurescaused by the operations of pressure application apparatus 16 andpressure buffering apparatus 18 and the maximum pressure Pmax andminimum pressure Pmin exceeds a predetermined value or not. If thereoccurs each error exceeding the predetermined value, the program goes tostep S5 where the amount of motion of the pressure application apparatus16 and pressure buffering apparatus 18 conforming to each value of themaximum pressure Pmax and minimum pressure Pmin is sampled, therebycorrecting a value stored in the main control apparatus 230.

Then in step S6, the programs in steps S3 to S6 are repeated until apredetermined culture time t elapses while when the predeterminedculture time t elapsed, the culture is completed, and the program goesto step S11.

In the culture mode at fixed pressure in step S7, the cell 5 or tissueis stimulated by a given pressure and the culture medium 3 is supplied.That is, in step S8, it is determined whether an error between apressure caused by the operation of the pressure application apparatus16 and pressure buffering apparatus 18 and a set pressure Ps exceeds apredetermined value or not. If there occurs an error exceeding thepredetermined value, the programs goes to step S9 where the amount ofmotion of the pressure application apparatus 16 and pressure bufferingapparatus 18 conforming to the set pressure Ps is sampled, therebycorrecting a value stored in the main control apparatus 230. In stepS10, when the predetermined culture time t elapses, the culture iscompleted, and the program goes to step S11.

Thereafter in step S11, a living body cell holding operation mode isperformed. Even if the culture of the cell 5 or tissue is completed ortissue is created, it is necessary to hold the cell 5 or tissue soundlyfor a while until the transfer of the cell 5 or tissue fortransplantation is started. In the living body cell holding operationmode, the culture medium 3 is supplied to the cell 5 to keep the livingbody cell in a sound state while maintaining the cell 5 at apredetermined temperature.

Then, in step S12, it is determined whether the living body cell istransplanted or not, namely, an operation stop instruction fortransplanting a tissue formed of the cell 5 is inputted or not, and thecirculation of the culture medium 3 and the control of temperature arestopped in response to the operation stopping instruction. Thereafter,the culture circuit unit 4 is detached from the culture apparatus 1 andthe cell 5 or tissue is transferred together with the culture circuitunit 4.

FIGS. 7, 8, and 9 show a set inputting operation in the initializationmode, wherein numerals a, b, c, d and e are used as connection symbolsof the divided flow charts, wherein the same or conformed letters of ato e extending over two pages are connecting portions.

In step S21, it is inputted that the cell 5 or tissue is cultivated inthe culture chamber 20 in a periodically pressurized state or at a fixedpressure. In step S22, when a pressure is varied periodically, theprogram goes to step S24 where “the variable pressure” is indicated. Onthe other hand, if the culture is performed under a fixed pressure, theprogram goes to step S23 where “fixed pressure” is indicated.

In step S25, a cycle T for varying a pressure is indicated. In step S26,it is determined whether the inputted cycle T is within execution ornot. If the cycle T is beyond execution, the program goes to step S27where “re-input of cycle T” is indicated and notified, then the programgoes to step S25 where the cycle T is re-inputted. If the cycle T iswithin execution, the program goes to step S28 where “cycle T” isindicated and it is stored in a memory of the main control apparatus230.

In step S29, holding time ti of the maximum pressure Pmax is inputted.In step S30, it is determined whether the holding time ti is within theoperation of the cycle T or not. If the holding time ti is beyond theoperation of the cycle T, the program goes to step S31 where “re-inputof holding time t₁” is indicated and notified, then the program goes tostep S29 where the holding time t₁ is re-inputted. If the holding timeti is within the operation of the cycle T, the program goes to step S32where “holding time t₁ of maximum pressure” is indicated and stored inthe memory of the main control apparatus 230.

In step S33, holding time t₂ of the minimum pressure Pmin is inputted.In step S34, it is determined whether the inputted holding time t2 iswithin the operation of the cycle T or not. If the holding time t₂ isbeyond the operation of the cycle T, the program goes to step S35 where“re-input of holding time t₂” is indicated and the program goes to stepS33 where the holding time t₂ is re-inputted. If the holding time t2 iswithin the operation of the cycle T, the program goes to step S36 where“holding time t₂ of minimum pressure” is indicated and stored in thememory of the main control apparatus 230.

In step S37, the inputted cycle T and the difference between times(t₁+t₂) are halved to operate pressure increasing/decreasing time t₃. Instep S38, it is determined whether the pressure increasing/decreasingtime t₃ is within operation or not. If the pressureincreasing/decreasing time t₃ is beyond operation, it is determined thatthe values of the cycle T, holding time t1 and holding time t₂ are notappropriate, and the program returns to step S25. If the pressureincreasing/decreasing time t₃ is within operation, the operated pressureincreasing/decreasing time t3 is stored in the memory of the maincontrol apparatus 230, and in step S39, “pressure increasing/decreasingtime t₃” is indicated. In step S40, it is inputted whether the speed ofmotion is varied or not when increasing or decreasing pressure. Theprogram goes to step S42 when the speed of motion is controlled in stepS41 while the program goes to step S46 when the speed of motion is notvaried.

In step S42, the amount of variation for controlling the speed of motionwhen increasing or decreasing a pressure is inputted. In step S43, it isdetermined whether the inputted amount of variation is operable or not.If inoperable, the program goes to step S44 where “re-input of theamount of variation in amount of increased/decreased pressure” isindicated, and the program goes to step S42 where it is re-inputted. Ifoperable, the program goes to step S45 where “amount ofincreased/decreased pressure” is indicated and it is stored in thememory of 230. At this time, the displacement of a pressure may beindicated on a simulation picture.

In step S46, the minimum pressure Pmin is inputted. In step S47, it isdetermined whether the minimum pressure Pmin is within execution or not.If the minimum pressure Pmin is beyond execution, the program goes tostep S48 where “re-input of minimum pressure Pmin” is indicated and theprogram goes to step S46 where the minimum pressure Pmin is re-inputted.On the other hand, if the minimum pressure Pmin is within execution, theprogram goes to step S49 where “minimum pressure Pmin” is indicated andstored in the memory of the main control apparatus 230.

In step S50, the maximum pressure Pmax is inputted. In step S51, it isdetermined whether the maximum pressure Pmax is within execution or not.If the maximum pressure Pmax is beyond execution, the program goes tostep S52 where “re-input of maximum pressure Pmax” is indicated and theprogram goes to step S50 where the maximum pressure Pmax is re-inputted.On the other hand, if the maximum pressure Pmax is within execution, theprogram goes to step S53 where “maximum pressure Pmax” is indicated andstored in the memory of the main control apparatus 230.

In step S54, a controlled temperature ct of the pressure chamber 60 isinputted. In step S55, it is determined whether the controlledtemperature ct is within execution or not. If controlled temperature ctis beyond execution, the program goes to step S56 where “re-input ofcontrolled temperature ct” is indicated and the program goes to step S54where the controlled temperature ct is re-inputted. If the controlledtemperature ct is within execution, the program goes to step S57 where“controlled temperature ct” is indicated and is stored in the memory ofthe main control apparatus 230.

In step S58, the amount of circulated flow f of the culture medium 3 inthe culture circuit unit 4 is inputted. In step S59, it is determinedwhether the amount of circulated flow f is within execution or not. Ifthe amount of circulated flow f is beyond the execution, the programgoes to step S60 or “re-input of the amount of circulated flow f” isindicated and notified, then the program goes to step S58 where theamount of circulated flow f is re-inputted. If the amount of circulatedflow f is within execution, the program goes to step S61 where “amountof circulated flow f” is indicated and is stored in the memory of themain control apparatus 230. In step S62, the operation time is inputted.

In step S63, “operation time” is indicated and is stored in the memoryof the main control apparatus 230.

Described hereinafter is the relation between the pressure applicationpiston 116 in the pressure application apparatus 16 and a pressureapplied to the cell 5 or tissue.

Since a force F is expressed by F=P×A, where A (cm²) is a sectional areaof the pressure application piston 116, P (kg/cm²) is a pressure and F(kgf) is a force, and the force F is further expressed by F=K×L₂, whereK (kgf/mm) is a spring constant of the pressure application spring 118and L₂(mm) is the amount of contraction of a spring, the followingequation is established. $\begin{matrix}\begin{matrix}{{K \times L_{2}} = {P \times A}} \\{L_{2} = {\left( {P \times A} \right)/K}}\end{matrix} & (1)\end{matrix}$

That is, when the pressure application piston 116 is moved, elasticityof the pressure application spring 118 acts on the pressure applicationpiston 116 so that the pressure application piston 116 compresses thewater 65 (for pressurizing) inside the pressure chamber 60. A pressureinside the pressure chamber 60 increases when the water 65 (forpressurizing) is compressed therein, and the increased pressure isdetected by the pressure sensor 112. The relation between thedisplacement of the pressure application piston 116, i.e., the amount ofmotion (mm) and the pressure P (kg/cm²) becomes e.g., as shown in FIG.10. In FIG. 10, L₁ is the amount of motion by the motor 122, L₂ is theamount of contraction of the pressure application spring 118, L₃ is theamount of motion of the pressure application piston 116 when thepressure application spring 118 is not used, L₄ is the amount of motionof the pressure application piston 116 caused by the shrinkage of mixedair, L₅ is the amount of motion of the pressure application piston 116caused by shrinkage of water and L₆ is the amount of motion of thepressure application piston 116 caused by the deformation of a vessel ofthe culture chamber 20 and pressure chamber 60. L₃ is the sum total ofL₄, L₅ and L₆, and L₁ is the sum total of L₂ and L₃. The relationbetween the amount of motion of the pressure application piston 116 inthe pressure application apparatus 16 and the value of pressure detectedby the pressure sensor 112 is stored in the memory of the main controlapparatus 230. The amount of motion of the pressure application piston116 caused by compression of air is described now.

Since a volume Va of air is expressed by Va=V/(Pa+1), where V (cm³) is avolume of air (at 1 atm.), Va (cm³) is the volume of air (whenpressurized) and 1×XV=(Pa+1) ×Va=constant is fixed while the amount ofmotion L₄ (mm) of the pressure application piston 116 caused bycompression of air is expressed as follows. $\begin{matrix}\begin{matrix}{L_{4} = {10 \times \left\{ {\left( {V - {Va}} \right)/A} \right\}}} \\{= {\left\lbrack {\left\{ {V - {V/\left( {{Pa} + 1} \right)}} \right\}/A} \right\rbrack \times 10}}\end{matrix} & (2)\end{matrix}$

Further, the amount of motion of the pressure application piston 116caused by compression of water and culture medium 3 becomes as follows.That is, since the amount of compression AW (cm3) of water and culturemedium 3 is expressed by ΔW=0.44×10⁻⁵×P×W where W (cm³) is the volume ofwater and culture medium 3 and the amount of compression of water (40°C.) is 0.44×10⁻⁵ (cm²/kg), and hence the amount of motion L₅ (mm) of thepressure application piston 116 caused by compression of water andculture medium 3 is expressed as follows. $\begin{matrix}\begin{matrix}{L_{5} = {\Delta \quad {W/A} \times 10}} \\{= {10 \times \left\{ {\left( {0.44 \times 10^{- 5} \times P \times W} \right)/A} \right\}}}\end{matrix} & (3)\end{matrix}$

The amount of contraction ΔWt is expressed by ΔWt=W×Ct where apparentcontraction percentage caused by the deformation of the pressure vessel22 and culture vessel 61 is Ct, and hence the amount of motion L₆ (mm)of the pressure application piston 116 caused by the deformation of thepressure vessel 22 is expressed as follows. $\begin{matrix}\begin{matrix}{L_{6} = {\left( {\Delta \quad {{Wt}/A}} \right) \times 10}} \\{= {10 \times \left\{ {\left( {W \times {Ct}} \right)/A} \right\}}}\end{matrix} & (4)\end{matrix}$

Accordingly, the total amount of motion of the pressure applicationpiston 116 becomes the value L₁ obtained by adding each amount ofmotions of the pressure application piston 116 in the expressions (1),(2), (3) and (4).

If a pressure applied to the buffer spring 154 is decreased at thepressure buffering apparatus 18 side, a pressure inside the culturechamber 20 exceeds a pressure applied to the pressure relief valve 26 sothat the pressure relief valve 26 is opened through which the culturemedium 3 passes, and hence a pressure at the culture chamber 20 side isdecreased. If a pressure application force of the buffer spring 154 isbalanced with a pressure at the culture medium 3 side, the pressure issettled. A pressure applied to the pressure relief valve 26 of thepressure buffering apparatus 18 is described now. A force F balancedwith the pressure P is expressed by F=P×B where a blocked area by thepressure relief valve 26 is B (cm²), a pressure is P (Kg/cm²), the forcebalanced with the pressure P is F (kgf), and the balanced force F isalso expressed by F=K×m and the amount of contraction m of the bufferspring 154 is expressed by m=P×B/K where a spring constant of the bufferspring 154 is K (kgf/mm) and the amount of shrinkage of the bufferspring 154 is m(mm). FIG. 11 shows a relation between a pressure appliedto the pressure relief valve 26 side, namely, the amount of motion ofthe actuator 156 side (amount of contraction of the buffer spring 154)and a pressure acting on the pressure relief valve 26, namely,controlled pressure. In FIG. 11, a line m₁ shows a case where a singlebuffer spring 154 is used, and a line m2 shows a case where twodifferent buffer springs are used.

Since the capacity of the medium supply apparatus 12 is small, it ispossible to substantially neglect the shrinkage of the culture medium 3,deformation of the vessel, and shrinkage of a gas, and so forth.Accordingly, the amount of motion I is expressed by I=V/C because theamount of supply of medium V(ml) of the medium supply piston 132 isexpressed by V=C×I where a sectional area of the medium supply piston132 is C (cm²) and the amount of motion is I (cm) so that the amount ofmotion is determined by the amount of supply of medium. If the amount ofmotion of the medium supply piston 132 of the medium supply apparatus 12is large, the medium supply piston 132 is returned to an originalposition after it is moved while if the amount of motion of the culturemedium 3 is small, the medium supply piston 132 is not returned to theoriginal position but it is moved further from that position during themedium supply operation, then it is returned to the original positionafter it is moved to an unmovable position. At this time, if a pressureis higher than an allowable value of a set decreased pressure, databetween the amount of the motion of the actuator 156 of the pressurerelief valve 26 and a pressure that is stored before the operation iscorrected to an original value.

FIGS. 12 (a) to 12 (d) show the manner of execution of the culture modeat a variable pressure to be executed in step S3 in FIG. 6, namely, FIG.12 (a) shows a state of pressure applied to the culture chamber 20 andFIGS. 12 (b) to 12 (d) show pressure application timings. That is, FIG.12 (a) shows the change or variation in pressure, FIG. 12 (b) shows anoperation timing of the pressure buffering apparatus 18, FIG. 12 (c)shows a pressure application timing of the pressure applicationapparatus 16, and FIG. 12 (d) shows a medium supply timing of theculture medium supply apparatus 6.

In the culture chamber 20, the increase and decrease of a pressure isrepeated between the maximum pressure Pmax and minimum pressure Pmin atthe cycle T. Each ti shows time for holding the maximum pressure Pmaxwhile each t₂ shows time for holding the minimum pressure Pmin, and eacht₃ shows operation time when increasing or decreasing a pressure. Thesemaximum pressure Pmax, minimum pressure Pmin, times t₁, t₂ , t₃ can bearbitrarily changed depending on the position in the living body wherethe cell 5 is cultivated in vitro. Further, it is possible to increaseor decrease a pressure by selecting appropriate numerical values basedon data relating to an age, a sex, a height, a weight respectively of aliving body, a position in the living body relating to the cell 5 to becultivated.

The pressure buffering apparatus 18 blocks the tube 50C by operating thepressure application apparatus 16 so that the pressure applicationapparatus 16 reaches a position where the maximum pressure Pmax isobtained at the maximum speed for time t₅ before the start of pressureapplication. Thereafter, the operation of the pressure applicationapparatus 16 is started upon elapse of delay time t₄ where a pressureapplication is performed to extend from the minimum pressure Pmin to themaximum pressure Pmax at the speed corresponding to time t₃.

After the maximum pressure Pmax is held for time t₁, the pressureapplication apparatus 16 is re-operated to start the decrease ofpressure to extend from the maximum pressure Pmax to minimum pressurePmin at the speed corresponding to time t₃. The pressure bufferingapparatus 18 is operated only for time t₇ while delayed by time t₆ afterthe pressure application apparatus 16 is operated so as to release theblocking of the tube 50C.

When the control of a pressure is started, a pressure is increased froma pressure close to zero pressure to the maximum pressure Pmax. At thistime, the pressure buffering apparatus 18 is moved to a blockingposition at the maximum speed while operating the pressure applicationapparatus 16 upon elapse of time t₉, and a pressure application isperformed for time t₈ until it reaches the maximum pressure Pmax at thespeed corresponding to time t₃.

The culture medium supply apparatus 6 operates for time t₁₂ upon elapseof time t₁₁, after holding the minimum pressure Pmin so as to supply theculture medium 3 to the culture chamber 20. It is possible toarbitrarily set the amount of supply of medium by changing the time t₁₂.

The medium supply piston 132 is moved backward for time t₁₄substantially equal to time t₁₂ upon elapse of the time t₁₃ after thesupply of medium. In this example, the medium supply is performed forthe holding time t₂ of the minimum pressure Pmin, but it can beperformed for a period of holding time t₁ of the maximum pressure Pmaxor for a period of pressure increasing and decreasing time t₃.

FIGS. 13 (a) to 13 (d) show the manner of execution of another culturemode at a variable pressure to be executed in step S3 in FIG. 6, namely,FIG. 13 (a) shows a state of pressure applied to the culture chamber 20and FIGS. 13 (b) to 13 (d) show pressure application timings. That is,FIG. 13 (a) shows the change or variation in pressure, FIG. 13 (b) showsan operation timing of the pressure buffering apparatus 18, FIG. 13 (c)shows a pressure application timing of the pressure applicationapparatus 16, and FIG. 13 (d) shows a medium supply timing of theculture medium supply apparatus 6.

In this example, a pressure pattern is obtained by controlling thevariation in pressure by varying a pressure increasing speed andpressure decreasing speed in quadratic function for pressureincreasing/decreasing time t₃ and the pressure pattern is outputted, sothat the pressure pattern applied to cartilage of knees when walking isreproduced because the variation in pressure is controlled. In thiscase, the pressure application apparatus 16 is varied in an operationspeed as shown in FIG. 13(C) for times t₁₅, t₁₆, t₁₇, and the control ofthe variation in pressure is added to the pressure application force fortime t₃. The other operations are the same as those shown in FIG. 12,and hence the explanation thereof is omitted.

Second Embodiment (FIGS. 14 to 21)

A method of and apparatus for cultivating a cell or tissue according toa second embodiment of the invention is described with reference toFIGS. 14 to 21, wherein FIG. 14 is a front view of the construction of aculture apparatus, FIG. 15 is a side view of the construction of aculture apparatus, FIG. 16 shows a main portion of the cultureapparatus, FIG. 17 shows a culture circuit unit 4, FIG. 18 shows a mainportion of the culture apparatus excluding the culture circuit unit 4,FIG. 19 shows a pressure application apparatus 16, FIG. 20 shows aculture medium supply apparatus 6, and FIG. 21 shows a pressurebuffering apparatus 18. Components which are the same as those in thefirst embodiment are denoted by the same reference numerals.

The culture apparatus is structured by a single housing 260. The housing260 is partitioned into a culture chamber 262, a machine chamber 264,and a control/power supply chamber 266. A culture box 42 is accommodatedin the culture chamber 262, and it has the same construction as that ofthe first embodiment except that the culture medium supply apparatus 6,a pressure application apparatus 16, the pressure buffering apparatus18, and so forth are structure by a single processing portion 268.

Doors 270, 272 which are independently opened or shut are providedrespectively in the culture chamber 262 and machine chamber 264. A watertank for pressurizing 68 is accommodated in the machine chamber 264together with the driving mechanism portions of the culture mediumsupply apparatus 6, pressure application apparatus 16 and pressurebuffering apparatus 18, wherein each of the actuators 120, 138, 156 issupported by the machine chamber 264 at the back side thereof with acommon fixing plate 269 as shown in FIG. 15. A water supply port 274 anda water discharge port 276 are respectively provided on a wall surfaceof the machine chamber 264. A control apparatus 40 and a power supplyapparatus are accommodated in the control/power supply chamber 266 andan indication apparatus 232 and a power supply switch 278 are providedon the front panel side of the control/power supply chamber 266.l

As shown in FIG. 16, the culture box 42 is accommodated in the culturechamber 262, and the culture circuit unit 4 and the processing portion268 are accommodated in the culture box 42. A processing unit 280 isdetachably provided in the processing portion 268 at the culture circuitunit 4 side as shown in FIGS. 17 and 18.

FIG. 19 shows the pressure application apparatus 16 including a culturevessel 61 and a pressure vessel 22 constituting a culture chamber 20. Inthis case, an actuator 120 of the pressure application apparatus 16comprises a housing 282 to which a ball screw 284 is attached, and amotor 122 coupled to the rear end of the ball screw 284 by a couplingjoint 286. A movable bed 288 that is moved back and forth by therotation of the ball screw 284 is provided on the ball screw 284, andtwo pressure application springs 118A, 118B which are overlapped witheach other are provided between the movable bed 288 and a support flange290 provided at the front end portion of the ball screw 284. That is,the two pressure application springs 118A, 118B are varied incompression state by the movable bed 288 that is moved in response tothe rotation of the ball screw 284 so that elasticity of the twopressure application springs 118A, 118B acts on the pressure applicationpiston 116 side. The actuator 120 may be formed of a belt or cam or thelike instead of the ball screw 284.

FIG. 20 shows the culture medium supply apparatus 6. The actuator 138comprises a housing 291 to which a ball screw 292 is attached and amotor 140 is coupled to the rear end portion of the ball screw 292 by acoupling joint 294. A movable bed 296 that is moved back and forth bythe rotation of the ball screw 292 is provided on the ball screw 292,and the front surface of a piston pressing board 298 attached to themovable bed 296 contacts the rear end of a medium supply piston 132.That is, when the movable bed 296 that is moved in response to therotation of the ball screw 292 caused by the motor 140 moves forward tocompress the pressure application spring 136, the medium supply piston132 moves forward so that the movable bed 296 is moved backward. As aresult, the compression of the pressure application spring 136 isreleased and the medium supply piston 132 moves backward by a restoringforce of the pressure application spring 136. The culture medium 3 canbe supplied when the medium supply piston 132 moves back and forth.

FIG. 21 shows the pressure buffering apparatus 18. In this case, theactuator 156 of the pressure buffering apparatus 18 comprises a housing300 to which a ball screw 302 is attached, a motor 158 coupled to therear end portion of the ball screw 302 by a coupling joint 304. Amovable bed 306 that is moved back and forth by the rotation of the ballscrew 302 is provided on the ball screw 302, and a plunger pressingboard 308 is attached to the movable bed 306 by way of buffer springs154A, 154B which are overlapped with each other, and the rear end of theplunger 152 of a pressure relief valve 26 contacts the front surface ofthe plunger pressing board 308. That is, when the movable bed 306 thatis moved in response to the rotation of the ball screw 302 caused by themotor 158 is moved forward, the plunger pressing board 308 moves forwardtogether with the buffer spring 154A, 154B so that the buffer spring154A, 154B are varied in compression state. That is, a valve body 150 ispressed by way of the buffer springs 154A, 154B which are in compressionstate so that the pressure relief valve 26 is held in a blocked state.This holding state is varied in response to the rotation of the ballscrew 302 and a compression state of the buffer springs 154A, 154Baccompanied by the rotation of the ball screw 302.

FIG. 22 shows a modification of the culture medium supply apparatus 6.Although the pressure application spring 136 is provided on the mediumsupply piston 132 in the culture medium supply apparatus 6 shown inFIGS. 2, 3 and 14, it may be possible to attach a connecting shaft 310to the movable bed 296 that is moved by the ball screw 292 of theactuator 138 while omitting the pressure application spring 136, and therear end of the medium supply piston 132 is coupled to the tip end ofthe coupling shaft 310 by way of fixing means such as a fixing pin 312.Even with such a construction, the medium supply piston 132 can be movedback and forth by the normal or reverse rotation of the ball screw 292.

Third Embodiment (FIG. 23)

A method of and apparatus for cultivating a cell or tissue according toa third embodiment of the invention is described with reference to FIG.23. Components which are the same as those in the first and secondembodiments are denoted by the same reference numerals.

In the third embodiment, pressurized air from a compressor, not shown isforced to act on the interior of the pressure chamber 60 formed of apressure vessel 22 of a pressure application apparatus 16 as indicatedby an arrow Pr through a conduit 67 on which a pressure regulator 314, apressure-increasing valve 316 and a needle valve 318 are provided, sothat the pressurized air in the pressure chamber 60 is dischargedthrough a collection conduit 102 having a needle valve 320 and apressure-decreasing valve 322. A valve 323 that is closable by therotation of an actuator 321 may be provided at the tube 50D side insteadof the valve 11 (FIG. 1) or pinch valve 162 (FIG. 2). Pressureapplication stimulation can be applied to the cell 5 by performing anoperation to intermittently block the valve 323 or an operation to applya pressure to the pressure transmitting film 64 while the pressurizedair acts on the pressure transmitting film 64. In this case, thepressure application stimulation can be varied by controlling thepressure-increasing valve 316 and pressure-decreasing valve 322 to beopened and shut. If such pressurized air is employed, the amount ofvariation in pressure every amount of motion per unit is made small at alow pressure while the amount of variation in pressure every amount ofmotion per unit is made large at a high pressure, so that unnecessaryvibration generated by a motor, an actuator and so forth can be absorbedwhen a pressure is applied to a cell or tissue, thereby enhancing theaccuracy of pressure application stimulation to be applied to the cellor tissue.

Fourth Embodiment (FIGS. 24 and 25)

A method of and apparatus for cultivating a cell or tissue according toa fourth embodiment of the invention is described with reference toFIGS. 24 and 25. Components which are the same as those in the first tothird embodiments are denoted by the same reference numerals.

A cell 5 to be cultivated is transplanted in a scaffold 7 formed of acollagen or the like, and it is stored in a culture chamber 20 everyscaffold 7. A culture medium 3 is supplied from a culture medium tank 49to the culture chamber 20 thorough a culture circuit unit 4. The culturecircuit unit 4 forms a closed circuit, and a pump 324 serving as themedium supply apparatus 12, a pressure sensor 326 and a pressurebuffering apparatus 18 are provided on the culture circuit unit 4. Apressure detected by the pressure sensor 326 is applied to a pressurecontroller 328, and a control output corresponding to the detectedpressure is applied to the pump 324 from the pressure controller 328.That is, a pressure P of the culture medium 3 is controlled to have afixed value.

The pressure buffering apparatus 18 comprises an actuator 156 attachedto a plunger 152 of a valve body 150 of a pressure relief valve 26 whichis inserted into a part of the culture circuit unit 4 by way of a bufferspring 154, and a motor 158 coupled to the actuator 156. The rotation ofthe motor 158, namely, a normal rotation, a reverse rotation, a stop ofrotation and an rpm are controlled by a control apparatus 40. That is,the rotation of the motor 158 is transmitted to a ball screw 302, and amovable bed 306 is moved back and forth in response to the direction ofrotation of the ball screw 302. Since a motion of the movable bed 306 istransmitted to the plunger 152 of the valve body 150 by way of thebuffer spring 154, a force to shut the valve body 150 is set by aposition of the movable bed 306 and a compression force of the bufferspring 154. When a pressure of the culture medium 3 caused by the pump324 exceeds a force to shut the valve body 150, the valve body 150 isopened so that the culture medium 3 passes through the pressure reliefvalve 26.

An air conduit 330 through which a gas such as oxygen or carbon dioxideis taken is provided in the culture medium tank 49, and a filter 332 forpreventing the entrance of bacteria, a foreign matter and so forth isprovided on the air conduit 330. That is, oxygen or carbon dioxide thatis taken in the culture medium tank 49 through the air conduit 330 istransmitted to the cell 5 inside the culture chamber 20 together withthe culture medium 3.

With such a construction, when the pump 324 is driven, the culturemedium 3 is supplied to the culture circuit unit 4 and flows in theculture chamber 20 so as to supply nutrition and a gas such as oxygen orcarbon dioxide that are needed for the cell 5. When the pressurebuffering apparatus 18 is driven, the culture circuit unit 4 is blockedso that a pressure inside the culture chamber 20 is increased by apressure applied from the pump 324 to the culture medium 3. A bufferingforce of the pressure buffering apparatus 18, namely, an arbitrarypressure value that is balanced with a pressure applied from the pump324 can be obtained by controlling a force for shutting the valve body150.

FIG. 25 shows a pressure application operation. When the pressurebuffering apparatus 18 is operated periodically, the maximum pressurePmax and the minimum pressure Pmin can be alternately applied to thecell 5. That is, in the cell 5, the maximum pressure Pmax is set fortime t₁, the minimum pressure Pmin is set for time t₂, and alsopressure-increase time t₃ and pressure-decrease time t₃ are set so thatthe circulation of the culture medium 3 under pressure is achieved likea living body and a growth environment is achieved like the living body.When an operation speed of the pressure buffering apparatus 18 iscontrolled, time ti, t₂, t₃ can be arbitrarily controlled, therebyachieving an optimal state corresponding to the characteristic of thecell 5 to be cultivated or the living body at a specific position. Themethod of and apparatus for cultivating a cell or tissue according tothe invention can obtain the following effects.

a. It is possible to cultivate a cell or tissue under an environmentmimicking the living body without being contaminated, and possible tocultivate the cell or tissue which is close to a tissue in a living bodyand easily fusible with a tissue in a living body.

b. It is possible to realize the culture of a cell or tissue which isideal and practical, corresponding to specific position of a living bodyto be restored, namely, close to a tissue in a living body and is easilyfusible with a tissue in a living body by holding a cell or tissue of aliving body at a specific culture position, setting the cell or tissueunder an environment mimicking the living body, supplying continuouslyor intermittently a culture medium to the cell or tissue, and applying apressure which is varied continuously, a pressure which is variedintermittently or a pressure which is varied periodically to the cell ortissue.

c. It is possible to cultivate a cell or tissue efficiently in aextremely stabilized state by holding a cell or tissue to be cultivatedin a suspending or non-suspending state in the culture medium.

d. It is possible to enhance the culture of a cell or tissue by holdinga cell or tissue in a suspending state in the culture medium by ahydro-gel or a scaffold.

e. It is possible to perform an efficient culture or cultivate the cellor tissue having high quality because the culture medium comprises oneof amino acids of various types, saccharides, salts and protein, or notless than two of materials selected therefrom or all of these materials.

f. It is possible to cultivate a cell or tissue which is easily fusiblewith a tissue in a living body because physiological conditions of theliving body at a specific position, an age, a height, a weight, a sex ofthe living body and other information inherent in the living body inaddition to the physiological conditions.

g. It is possible to realize the control of an environment close to aliving body, and possible to contribute to the culture of a cell ortissue which is close to a tissue in a living body and is easily fusiblewith a tissue in the living body because a living body environment isset by supplying and controlling a nitrogen gas, an oxygen gas, a carbondioxide gas and by setting and controlling a temperature and a humidity.

h. It is possible to form a cell or tissue which is ideal and practicalby applying a pressure to the cell or tissue, corresponding to a livingbody at a specific position to be restored.

i. It is possible to realize ideal physical stimulation by selecting orcombining modes of a pressure pattern which is varied continuously,intermittently or periodically, which affects metabolism function, celldivision cycle, concentration gradient or dispersion of living bodystimulation, thereby enhancing the culture.

j. It is possible to protect or prevent a cell or tissue which isintercepted from an open air from being contaminated by bacteria tocultivate a high quality cell because a culture unit allows a cell ortissue to be cultivated to be accommodated in a culture chamber andsupplies culture medium needed for the cell or tissue which isintercepted from an open air. Further, a desired pressure by pressureapplication means as well as a hydraulic pressure by culture medium andphysical stimulation by flow of culture medium are applied to a cell ortissue, the cell or tissue susceptible to metabolism function, celldivision cycle, concentration gradient or dispersion of living bodystimulation, thereby enhancing culture of the cell or tissue. Stillfurther, since the mode of supply of culture medium to a cell or tissueis arbitrarily set by culture medium supply means so that culture mediumcan be supplied intermittently or continuously, thereby enhancingculture by a variety of physical stimulation.

k. It is possible to mimic the living body, set a desired environment,and perform an efficient culture by arbitrarily controlling the pressureapplication means or culture medium supply means using control means,and performing various program controls such as a feed back control orfeed forward control by the control means such as a computer.

l. The manner of applying a pressure, namely, a pressure pattern is setin response to the cell or tissue to be cultivated, thereby performingmore efficient culture.

m. It is possible to perform efficient culture by setting a pressurepattern in all modes, and selecting or combining thereof.

n. It is possible to protect or prevent the cell or tissue from beingcontaminated by bacteria and so forth during the motion thereof toenhance a reliability such as restoration of a living body because theculture unit having a culture chamber for accommodating the cultivatedcell or tissue is independent of and detachable from a culture apparatusbody, so as to move the cell or tissue together with the culture unitthat is separated from an open air.

o. It is possible to set a culture environment by the supply of adesired gas because a hermetically sealed space serving as a culturespace is intercepted from an open air, and also protect or prevent thecell or tissue from being contaminated by the open air.

p. It is possible to apply a gas to the cell or tissue by supplying agas such as a nitrogen gas, an oxygen gas, a carbon dioxide gas andproviding gas absorption means in a culture unit and possible to mimic aliving body environment by supplying and controlling the gas.

q. It is possible to mimic a living body environment and provide adesired culture space by filling a nitrogen gas, an oxygen gas, a carbondioxide gas into the culture space formed by a hermetically sealedspace.

r. It is possible to prevent culture medium from being contaminated byproviding a culture medium tank for supplying the culture medium to theculture unit or circulating the culture medium and installing it in ahermetically sealed space that is intercepted from the open air.

s. It is possible to apply pressure application stimulation to a cell ortissue accommodated in a culture chamber in a state wherein it isintercepted from an open air, and to realize desired pressureapplication stimulation such as stimulation mimicking a living bodyenvironment by providing a pressure transmitting film.

t. It is possible to realize physical stimulation close to a living bodyenvironment and to enhance the culture of a cell or tissue by regulatinga pressure by pressure buffering means when a part of a culture unit ispressurized.

u. It is possible to realize desired pressure application stimulationand to mimic a living body environment with high accuracy by usingeither of a hydraulic pressure, an oil pressure or an air pressure aspressure forming means.

v. It is possible to supply and circulate a culture medium efficientlyto a culture unit if the culture medium means comprises a medium supplyapparatus for pressuring the culture medium that is taken in the mediumsupply chamber and possible to set the amount of desired supply ofmedium by controlling the amount of applied pressure.

w. It is possible to apply ideal pressure application stimulation to thecell or tissue because a pressure to be applied to the culture medium isbuffered, and possible to control the culture medium in an idealpressure state, when using the pressure relief valve, withoutcontaminating the culture medium if the pressure of the culture mediumis decreased by opening the pressure relief valve while controlling thepressure relief valve.

x. It is possible to provide a culture space conforming to a living bodyenvironment by controlling a temperature and a humidity of ahermetically sealed space in which the culture unit is accommodated.

y. It is possible to mimic a living body environment acoustically byusing a sound producing unit together because a living body receivesacoustic stimulation from the outside, and possible to inject the cellor tissue to be cultivated in a culture chamber by use of a super-soundwave together with high reliability.

z. It is possible to mimic a living body environment to contribute tothe enhancement of culture of a cell or tissue by controlling theconcentration of a gas to be supplied to a hermetically sealed space bycontrolling means.

Although the construction, function and effect of the method of andapparatus for cultivating a cell or tissue according to the first tofourth embodiments of the invention are described, the invention is notlimited to these embodiments, and the invention includes allconstructions such as various constructions, modifications, and so forthwhich can be expected or conjectured based on the object and theembodiments of the invention.

What is claimed is:
 1. A method of cultivating a cell or tissue of aliving body to be cultivated inside a culture chamber comprising thesteps of: forming a sealed space while isolated from the outside intowhich space gas is supplied, and in which space temperature and humidityconditions are set to maintain an environment necessary for sustaininglife; forming a culture circuit unit which is separable from the sealedspace; the culture circuit unit comprising a culture fluid bath and theculture chamber which are installed therein and connected to each otherby a tube to form a culture circuit through which circuit culture fluidcan be circulated from the culture fluid bath to the culture chamber;supplying the culture fluid continuously, intermittently or periodicallyor by the combination thereof from the culture fluid both so as tocirculate the culture fluid in the culture chamber through the culturecircuit; rendering the cell or tissue in the culture chamber to be heldin a suspending or non-suspending state by holding means in the culturefluid within the culture chamber; and forming a gas absorption portionin the tube of the culture circuit at a front stage of the culturechamber separately from the culture fluid bath, through which the gas inthe sealed space permeates and rendering the gas to be absorbed in theculture fluid which flows toward the culture chamber through the tube;wherein when the cell or tissue held by the holding means grows, theholding means is absorbed in the cell or tissue and vanishes.
 2. Themethod of cultivating a cell or tissue according to claim 1, wherein thecell or tissue is held in the culture fluid by a hydro-gel.
 3. Themethod of cultivating a cell or tissue according to claim 1, wherein theculture fluid comprises at least one of the group consisting of aminoacids, saccharides, salts and proteins.